Manufacture and purification of mycophenolic acid

ABSTRACT

The present invention provides an improved method for the manufacture of Mycophenolic acid by solid substrate fermentation of  Pencillium brevi-campactum , in a contained bioreactor under optimal fermentation parameters with its subsequent purification steps.

FIELD OF THE INVENTION

The present invention relates to an improved method for the manufactureand purification of Mycophenolic acid (MPA).

BACKGROUND OF THE INVENTION

Mycophenolic acid (MPA) was initially isolated from a culture ofPenicillium (B. Gosio, Riv. Igiene Sanita Pub. Ann, 7, 825–869, 1896).Its value as an immunomodulator was realized much later. Morpholinoester of mycophenolic acid is used as a prodrug in pharmaceuticalcomposition for treatment of rheumatoid arthritis, psoriasis and inprevention of tissue rejection in organ transplant patients.

Mycophenolic acid is produced by aerobic fermentation of severalPenicillium species. It has a broad spectrum of activity like antitumoractivity, antiviral, antipsoriatic, immunosuppressive andanti-inflammatory activity. It also exhibits antibacterial andantifungal activities. It is tolerable in large doses and has minimalside effects. It inhibits inosine monophosphate dehydrogenase which isan important enzyme in de novo synthesis of inosine monophosphate, aprecursor of purines. MPA also inhibits proliferation of lymphocytesthat are responsible for immune response. This immuno repressory effectof mycophenolic acid has been important in treatment of organ rejectionafter organ transplant surgery.

There is a continuous need to find improved process for production ofmycophenolic acid that will be commercially viable. It has been shownthat mutants Penicillium brevi-compactum resistant to polyeneantibiotics, HMG CoA reductase inhibitors, methyl viologen andsurfactants produce more MPA than the parent strain (U.S. Pat. No.4,452,891).

The proposed biosynthetic pathway of mycophenolic acid productioninvolves steroid biosynthetic pathway as well as polyketide biosyntheticpathway. It is a condensation product of a tetraketide and geranylpyrophosphate, wherein geranyl moiety is cleaved after condensation andsubsequent o-methylation in the tetraketide ring yields mycophenolicacid (Muth and Nash, Antimicrobial Agents and Chemotherapy, 8, 321–327,1896). The biosynthetic pathway was studied in P. stoloniferum.

Penicillium brevi-compactum strain has been used in submergedfermentation where it produces 2.4 mg/ml at 27° C. in 6 days on shakingand gives 3.6 mg/ml at 27° C. in 14 days without shaking (U.S. Pat. No.4,452,891). In solid substrate fermentation (SSF) it is reported toproduce 3286 mg per Kg of wheat bran (Sadhukhan et al, J. Ind.Microbiol. Biotechnol. 22, 33–38, (1999). Regarding the economics ofusing a producing strain, the described volume-time-yields as anindustrial process are not economically attractive.

The object of the present invention is to provide an improved method forthe production of mycophenolic acid by solid substrate fermentation in anovel bioreactor ‘PLAFRACTOR’ and its subsequent purification.

To achieve the said objective this invention provides an improved methodfor the manufacture of Mycophenolic acid comprising:

-   -   loading a contained bioreactor with solid substrate matrix and        sterilizing it,    -   mixing the said sterilized solid substrate matrix with        Penicillium brevi-compactum,    -   adding 5–20% of glycerol, if desired,    -   incubating the said inoculated solid substrate matrix for 4–7        days at 20–35° C.,    -   extracting the fermented solid substrate matrix with an organic        solvent,    -   concentrating the organic solvent extract,    -   crystallizing mycophenolic acid by adjusting the pH to about 2.0        with an inorganic acid and allowing to stand for about 3 hours,        followed by filtration using a filter aid,    -   dissolving the filter aid cake in a water immiscible organic        solvent and treating with alumina,    -   filtering the water immiscible organic solvent followed by        concentration of the organic layer by distillation,    -   dissolving the concentrate obtained in an alcohol,    -   dispersing the alcoholic solution in water and filtering to get        the crude crystals,    -   dissolving the crude crystals thus obtained in an organic        solvent,    -   adding another organic solvent to the previous step solution and        chilling to 4 to −20° C. to get pure mycophenolic acid crystals.

The Penicillium brevi-compactum used is in the form of spore suspensionor in mycelial form.

The solid substrate matrix is selected from wheat bran, rice bran, ragiflour, soya flour, cotton seed flour, wheat flour, rice flour, ricehusk.

The solid substrate matrix is a mixture of two or more solid substancesselected from wheat bran, rice bran, ragi flour, soya flour, cotton seedflour, wheat flour, rice flour, rice husk.

The said contained bioreactor allows solid state fermentation to becarried out in a manner such that the fermentation micro-organisms andthe fermentation products produced are kept isolated from the outsideenvironment during the course of fermentation. The said containedbioreactor is “PLAFRACTOR”.

The organic solvent used for extraction is selected from acetone,ethanol, toluene, benzene or ethyl acetate.

The filter aid is selected from celite, perlite or alumina.

The solvent used to dissolve the filter aid cake is selected fromcyclohexane, toluene, benzene, ethyl acetate or butyl acetate.

The alcohol used for crystallization is selected from methanol, ethanolor iso-propanol.

The inorganic acid used for adjusting pH is sulphuric acid and theconcentration of the organic layer is carried out by azeotropicdistillation.

The present invention uses Penicillium brevi-compactum. The colony ofthis isolate was comparatively fast growing and the aerial mycelium wascottony and white. The microbial culture sporulated showing greencoloration on third day.

The bioreactor used for the solid substrate fermentation is ourinvention and is described in our PCT publication no. WO 00/29544. Thesaid bioreactor is modular in nature and carries out all of theprocesses of solid substrate fermentation in a single containedenvironment. The modular construction of the bioreactor providesmultiple modules stacked on top of one another, each with a baseconnected to frame for holding the solid medium in isolation from theexterior environment. The construction of the bioreactor allows solidsubstrate fermentation to be carried out in a manner such that thefermenting microorganisms and the fermentation products it produces arekept isolated from the outside environment during the course of thefermentation. This containment of the fermentation process is ofsignificant importance when working with microbial metabolites, whichare cytotoxic in nature e.g. Cyclosporin, mycophenolic acid.

The said bioreactor operates in a contained manner and is capable ofsterilizing the solid state fermentation media, cooling it to therequired temperature, fermenting at the desired set conditions, in situextraction of the end product, recovery of the solvents and post harveststerilization.

An important aspect of the bioreactor is a mechanism of heat removalresulting in stringent temperature control of the fermentation process.In comparison, maintaining a constant temperature of growth in solidsubstrate fermentation using tray cultures is not efficient. The baseplate of the bioreactor has multiple channels called noncommunicatingchannels that carry heating and cooling fluids sandwiched between twosheets. Heat is transferred to and from the modules by conduction. Inthis way the temperature of the module is precisely maintained to meetthe specific requirement of different microorganisms.

The base of the module contains a second set of channels, thecommunicating channels to deliver sterile air as supply of oxygen intothe solid substrate bed for optimum growth of organism. Moisture lossbecause of passage of sterile air is significantly reduced by regularlyreversing the direction of airflow every few hours. Using this,homogeneity in moisture content is maintained throughout the bioreactor.These aspects provide ample convenience over previous SSF methodologiesthat require multiple manipulations at each step of the fermentationprocess.

The invention will now be described with reference to the followingexamples:

EXAMPLE 1

A single spore isolate of Penicillium brevi-compactum was used. Theorganism was subcultured on a fresh MEA (Malt Extract Agar) slant andincubated at 24° C. After 5 days, the sporulated slant was suspended in10 ml of water containing 0.01% tween 80. 500 μl of this sporesuspension were spread on a fresh plate containing MEA. The plate wasallowed to grow for 5 days. After 5 days the spores were scraped fromthe plate with a sterile loop and suspended in sterile distilled water.This spore suspension, devoid of mycelial fragments was used as theinoculum. 15 Kg of wheat bran was loaded on the bioreactor ofapproximately 22600 cm² of plate area. The bioreactor was sterilized bysending steam simultaneously into the communicating and thenoncommunicating channels to heat the bioreactor and its contents to atemperature of 121° C. for 90 minutes. The steam pressure was releasedand simultaneously sterile air was sent into the communicating channelswhile cooling water at approximately 25° C. was sent into thenoncommunicating channels.

The master seed for inoculation of culture was a 10⁶ spores/mlsuspension of Penicillium brevi-compactum in 14 L of sterilizeddistilled water containing 20% glycerol. This was used to inoculate thesterilized wheat bran so that the final moisture after inoculation was60%. The inoculum was mixed thoroughly with the sterilized bran. Sterileairflow at a rate of 20 Lpm on the first day, 40 Lpm on second and thirdday and 20 Lpm on fourth and fifth day were sent into the bioreactorcontinuously. The temperature was controlled at 25° C. for all 5 days byconductive heating and cooling. The Mycophenolic acid production titreswere assayed following extraction using the HPLC.

EXAMPLE 2

The Bioreactor was sterilized and inoculated as in Example 1. In thisexperiment, the temperature was maintained at 30° C. for all 5 days. TheMycophenolic acid production titres were assayed following extractionusing the HPLC.

EXAMPLE 3

5 Kg. fermented wheat bran obtained from example 1 was then extracted byusing 10 L of acetone and the extract was collected, analyzed and takenfor further processing. The extraction efficiency of acetone was foundto be 98%, as quantitated by HPLC.

EXAMPLE 4

The extract obtained from Example 3 was concentrated by azeotropicdistillation to remove acetone, leaving behind 1.5 L of aqueous residue.The pH of this aqueous residue was adjusted to 2.0 with concentratedH₂SO₄ and allowed to stand at 10° C. After 4 hours large needle shapedcrystals of Mycophenolic acid were found floating at the surface of theliquid. These crystals were separated by filtration through a celitebed. Recovery of mycophenolic acid by this crystallization was found tobe 100%. Crystals trapped on the celite bed were re-dissolved completelyin 2 L of ethyl acetate. Ethyl acetate layer was separated and treatedwith alumina to remove colour. Alumina was removed by filtration andethyl acetate layer was concentrated by distillation to leave behindlight brown coloured crystals of mycophenolic acid. These crystals werefurther dissolved in methanol and dispersing the methanolic solution inwater to obtain white crystals of mycophenolic acid. These crystalsobtained from aqueous methanol were dissolved in 10 parts of acetone. Tothis acetone solution equivalent amount of hexane was added and themixture was chilled to 10° C. The crystals of mycophenolic acid wereseparated by filtration and dried. The crystals thus obtained were ofacceptable pharmaceutical grade.

The present invention has the following advantages over the otherreported methods:

-   -   (i) Fermentation in a bioreactor, which is fully contained as a        result assuring full safety for the cytotoxic fermentation        products like mycophenolic acid.    -   (ii) In situ extraction of the fermented end product.    -   (iii) Less fermentation time making the process economically        attractive.    -   (iv) Fewer steps for the isolation and purification to get the        pure product, thus saving processing time and additional        expenses.

1. A method for the manufacture of Mycophenolic acid comprising: loadinga contained bioreactor with solid substrate matrix and sterilizing it,mixing the said sterilized solid substrate matrix with Penicilliumbrevi-compactum, adding 5–20% of glycerol, if desired, incubating thesaid inoculated solid substrate matrix for 4–7 days at 20–35° C.,extracting the fermented solid substrate matrix with an organic solvent,concentrating the organic solvent extract, crystallizing mycophenolicacid by adjusting the pH to 2.0 to 5.0 with an inorganic acid andallowing to stand for 3 to 10 hours, followed by filtration using afilter aid, dissolving the filter aid cake in a water immiscible organicsolvent and treating with alumina, filtering the water immiscibleorganic solvent followed by concentration of the organic layer bydistillation, dissolving the concentrate obtained in an alcohol,dispersing the alcoholic solution in water and filtering to get thecrude crystals, dissolving the crude crystals thus obtained in anorganic solvent, adding another organic solvent to the previous stepsolution and chilling to 4 to −20° C. to get pure mycophenolic acidcrystals.
 2. A method as claimed in claim 1 wherein Penicilliumbrevi-compactum used is in the form of spore suspension or in mycelialform.
 3. A method as claimed in claim 1 wherein the solid substratematrix is selected from wheat bran, rice bran, ragi flour, soya flour,cotton seed flour, wheat flour, rice flour, nice husk.
 4. A method asclaimed in claim 1 wherein the solid substrate matrix is a mixture oftwo or more solid substances selected from wheat bran, rice bran, ragiflour, soya flour, cotton seed flour, wheat flour, rice flour, ricehusk.
 5. A method as claimed in claim 1 wherein the organic solvent usedfor extraction is selected from acetone, methanol, toluene, benzene orethyl acetate.
 6. A method as claimed in claim 1 wherein the filter aidis selected from celite, perlite or alumina.
 7. A method as claimed inclaim 1 wherein the solvent used to dissolve the filter aid cake isselected from cyclohexane, toluene, benzene, ethyl acetate or butylacetate.
 8. A method as claimed in claim 1 wherein the alcohol used forcrystallization is selected from methanol, ethanol or iso-propanol.
 9. Amethod as claimed in claim 1 wherein the inorganic acid used foradjusting pH is sulphuric acid.
 10. A method as claimed in claim 1wherein the concentration of the organic layer is carried out byazeotropic distillation.